Project Summary/Abstract The purpose of this study is to determine whether genetic variants associated with AD risk in the SORL1 gene and other endocytic genes lead to endosomal network dysfunction and cellular AD phenotypes in human neurons. Endosomal abnormalities are documented in post-mortem AD brain tissue and multiple endocytic regulatory genes are associated with increased AD risk in population studies. SORL1 is a vesicular trafficking gene that functions in transporting cargo between endosomes, Golgi and the plasma membrane. SORL1 plays an integral in trafficking amyloid beta and the amyloid precursor protein through the endocytic network and loss of SORL1 is documented in AD brain tissue, possibly contributing to senile plaque formation. This pathway represents a novel avenue for therapeutic development in AD. Our previous studies have used human induced pluripotent stem cell (hiPSC)-derived neurons (hiPSC-Ns) to show that deficiencies in SORL1 expression induction are correlated with the presence of AD-associated variants in non-coding regions of SORL1. In this work, we hypothesize that risk variants in endosomal network genes predicts cellular endocytic and AD relevant phenotypes. To test this hypothesis, we will leverage our long-standing expertise in hiPSC-derived neuronal differentiations with our newly developed methods to generate hiPSC-neurons and directly transdifferentiated neurons from post-mortem AD tissue to test i) whether AD associated variants lead to loss of function of SORL1 resulting in endosomal and AD-relevant phenotypes; ii) whether cellular age exacerbates these phenotypes; and iii) whether a cumulative burden of AD risk variants in the endocytic pathway predicts endocytic phenotypes. We have identified AD patients with SORL1 coding variants and have obtained fibroblasts from these patients for hiPSC-generation. We will use CRISPR/Cas9 gene editing to correct the variants in patient cells and introduce the variants in control cells, generating an allelic series of cell lines that will include one or two copies of the variant allele as well as SORL1 knock-out cell lines. We will differentiate neurons from these lines and assay defined phenotypes of endosomal and AD pathology: Enlarged endosome size, decreased endocytic recycling, increased A? peptide secretion and increased Tau phosphorylation. Furthermore, we will generate induced neurons (iNs) from patient and control fibroblasts by direct conversion to test whether endocytic phenotypes are enhanced when cellular age is maintained. Finally, we will derive hiPSC-Ns and iNs from cases with autopsy confirmed AD and high risk burdens of AD-associated SNPs in endocytic genes. We will perform our endosomal assays and generate phenotypic groups. This work is significant in that it will investigate a functional genotype- phenotype relationship of genetic variants in the endosomal network, which is known to be disrupted early in AD pathogenesis. Investigating this driver of disease pathogenesis and how it relates to human genetic background is critical in the development of new and precision treatments for AD.